The studies outlined in this proposal are a continuation of our effort towards achieving the long-term goal of developing novel approaches to selective inhibition of matrix metalloproteinase (MMP) activity during cancer progression. In the previous funding period we focused on the gelatinases, MMP-2 and MMP-9 and we reported the successful development and characterization of the first mechanism-based gelatinase inhibitor, SB-3CT, and demonstrated its effectiveness in various tumor models in mice. Our approach to selective MMP inhibitor design has provided a point of departure for the field. We now propose to develop this approach for effective and selective inhibition of membrane-type MMPs (MT-MMP), a subfamily of membrane-anchored MMPs that has received considerable attention in the last five years due to their key roles in cancer cell invasion. This new information highlights the MT-MMPs as promising therapeutic targets that deserve further exploration. However, to achieve this goal there is critical need for effective and selective MT-MMP inhibitors, which are currently missing. Preliminary evidence from our laboratory demonstrates the development of new mechanism-based MT1-MMP inhibitors with great potential. Also, a novel inhibitor library has been conceived, which will permit discovery of more selective mechanism-based inhibitors for MT-MMPs based on the molecular template of SB-3CT. Finally, to aid in the detection of active MMPs in cancer tissues, we have synthesized and tested an inhibitor-tethered resin, which can be used to ascertain the level of active protease during cancer progression and inhibitor treatment. Here we propose to continue our effort using a comprehensive approach of biochemical and biological studies for identification and inhibition of MT-MMPs. Specifically, we will (1) Identify selective mechanism-based inhibitors for transmembrane MT-MMPs, (2) Validate the inhibitory properties of the MT-MMP inhibitors in cellular systems of MT-MMP-dependent pericellular proteolysis, (3) Test the MT-MMP inhibitors in animal models of cancer, and (4) Monitor active forms of the MT-MMP/gelatinase axis in tumor tissues as a function of tumor development and MT-MMP inhibitor treatment. The results of the present application will contribute to the collective endeavor of developing novel and rational approaches for controlling unregulated proteolysis in cancer tissues mediated by MT-MMPs.